Breast cancer is the second leading cause of cancer-related death and second most frequently diagnosed cancer in women in the United States1. Breast conserving surgery with radiation has emerged as the preferred method for treating breast cancer while preserving breast tissue and without compromising survival rates. Local recurrence following partial mastectomy is strongly correlated to the surgical margin2-4, but despite evaluation using a combination of intraoperative examination and post-operative histopathology, 40% of patients require surgical re-excision as a result of positive or close margins5, 6. Two-photon microscopy (TPM) has demonstrated significant resolution, molecular specificity, and light efficiency advantages over conventional white-light and fluorescence microscopes for imaging endogenous and exogenous contrast both in vivo and ex vivo7-9. However, scattering tissue severely degrades two-photon signals and resolution, limiting the fundamental imaging depth of TPM to ~100 <m10-13. Optical coherence tomography (OCT) is a method of utilizing low-coherence interferometry to detect the scattering properties of tissue down to penetration depths of ~1-2 mm14. The high detection sensitivity and coherence-gating of out-of-focus light of OCT has also been shown to enhance optical sectioning through highly scattering media when used in conjunction with confocal microscopy (optical coherence microscopy, OCM)15. OCM has recently demonstrated the capacity for optical biopsy by providing cellular-resolution images of freshly-excised bulk breast pathology specimens16. The current proposal emphasizes the development of a wavefront corrected multimodal OCM-TPM system to rapidly assess breast tumor margins. OCM-TPM will combine the advantages of complimentary contrast, depth penetration, and molecular-sensitivity of the respective modalities. A novel 10 fs pulsed laser source will be used to enhance two-photon fluorescence and allow for exquisite axial sectioning resolution using OCM. Further, the scattering wavefront will be compensated by using adaptive correction and feedback algorithms to improve the signal intensity and resolution of both OCM and TPM in scattering tissue. Concurrently, we propose to identify optimal intrinsic and extrinsic biomarkers and surrogates for optical evaluation of breast tumor margins, including nuclear size and shape and collagen orientation, as well as exogenous contrast sensitive to cytokeratins, hormone receptors, and gene amplification. The utility of OCM-TPM, relative to standard immunohistochemistry, will be assessed in a clinical setting to characterize cancer detection sensitivity and specificity. The proposed multimodal non-destructive optical imaging tool will allow for rapid intraoperative assessment of surgical margins as a means of reducing the morbidity and mortality associated with breast cancer.